NPN uptake assay

The 1-N-phenylnaphthylamine (NPN) uptake assay was performed as described previously (Martinez De Tejada and Moriyon, 1993). A. pleuropneumoniae strains MD12, ΔsapA, and PΔsapA were grown to OD₆₀₀ 0.6 and harvested by centrifugation at 2,500 g for 15 min. Pellets were washed three times and resuspended in 5 mM HEPES buffer (pH 7.2) containing 10 μM NPN (Sigma–Aldrich, USA). NPN uptake into the A. pleuropneumoniae ΔvacJ mutant was used as a positive control, as vacJ encodes VacJ lipoprotein and the membrane permeability of this mutant was increased as described previously (Xie et al., 2016). Fluorescence was measured using the EnVision Multilabel Reader (PerkinElmer, UK), with emission at 420 nm and excitation at 350 nm.